Hyposmotic stress causes ATP release and stimulates Na,K-ATPase activity in porcine lens

Purinergic receptors in lens epithelium suggest lens function can be altered by chemical signals from aqueous humor or the lens itself. Here we show release of ATP by intact porcine lenses exposed to hyposmotic solution (200mOsm). 18a-glycyrrhetinic acid (AGA) added together with probenecid eliminated the ATP increase. N-ethylmaleimide (200 mu M), an exocytotic inhibitor, had no significant effect on ATP increase. Lenses exposed to hyposmotic solution displayed a similar to 400% increase of propidium iodide (PI) entry into the epithelium. The increased ability of PI (MW 668) to enter the epithelium suggests possible opening of connexin and/or pannexin hemichannels. This is consistent with detection of connexin 43, connexin 50, and pannexin 1 in the epithelium and the ability of AGA+probenecid to prevent ATP release. Na,K-ATPase activity doubled in the epithelium of lenses exposed to hyposmotic solution. The increase of Na,K-ATPase activity did not occur when apyrase was used to prevent extracellular ATP accumulation or when AGA+probenecid prevented ATP release. The increase of Na,K-ATPase activity was inhibited by the purinergic P2 antagonist reactive blue-2 and pertussis toxin, a G-protein inhibitor, but not by the P2X antagonist PPADS. Hyposmotic solution activated Src family kinase (SFK) in the epithelium, judged by Western blot. The SFK inhibitor PP2 abolished both SFK activation and the Na,K-ATPase activity increase. In summary, hyposmotic shock-induced ATP release is sufficient to activate a purinergic receptor- and SFK-dependent mechanism that stimulates Na,K-ATPase activity. The responses might signify an autoregulatory loop initiated by mechanical stress or osmotic swelling. J. Cell. Physiol. 227: 1428-1437, 2012. (C) 2011 Wiley Periodicals, Inc.